Truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi strains Karp, Kato and Gilliam and its use in antibody based detection assays and vaccines

ABSTRACT

A recombinant, refolded non-fusion polypeptide expressed from a truncated r56 gene of the causative agent of scrub typhus,  Orientia tsutsugamushi  for the Karp, Kato and Gilliam strains has been produced. The invention is useful for detecting prior exposure to scrub typhus, screening for and/or identification of at least one infectious strain-similarity (i.e. a Karp-like, Kato-like or Gilliam-like strain) based on its strength of reaction toward a truncated protein and as a component in vaccine formulations and production of immune globulins for passive prophylaxis and immunity in subjects.

CROSS-REFERENCE

This is a Continuation-In-Part Application of application Ser. No.09/218,425 filed Dec. 22, 1998 (now U.S. Pat. No. 6,482,415) whichclaims priority from U.S. Provisional Application No. 60/068,732 filedon Dec. 24, 1997 and U.S. Provisional No. 60/283,373 filed on Apr. 13,2001.

BACKGROUND OF THE INVENTION

Some of the most dreadful pandemics in the history of mankind resultfrom the rapid spread of infectious diseases caused by virulentpathogenic organisms. These disease states are often accompanied byother opportunistic infections (viral, protozoal, bacterial orparasitic) and/or diseases due to the compromised immune system ofinfected patients.

There is new evidence that new epidemics are emerging throughout theindustrialized, developing and transitional countries of the world.

Today, the rates of reported cases of diseases are increasing inexponential proportions and clinical treatments currently availablerepresent only marginal improvements in the management of health care inthis area. The rapid increase in the number of infectious diseasesranges from alarming to out of control. Unless improved treatments arefound the future outlook for the state of the world's health is dismal.The scientific community throughout the world is mindful of thelong-felt need for effective ways to

(1) substantially reduce, eliminate, neutralize and/or kill virulentpathogenic organisms or agents,

(2) inhibit the proliferation of rapidly replicating abnormal (infectedor altered) cells caused by pathogenic organisms, or agents such as avirus, bacteria, fungus, venom, pollen, protozoal, and mixtures thereofand

(3) identify effective vaccines, preventive (prophylactic) andtherapeutic treatments for patients, including humans. In response tothe need to alleviate suffering and provide comfort to human life, thescientific community is searching for effective means to inhibit thegrowth of rapidly proliferating abnormal mammalian cells caused bypathogenic organisms within the genus Rickettsia, such as O.tsutsugamushi alone and/or in combination with others.

Scrub typhus, also referred to as chigger-borne rickettsiosis,mite-borne typhus, Japanese river fever, tropical or rural typhus ortsutsugamushi disease is an acute, febrile disease caused by infectionwith Orientia (formerly Rickettsia) tsutsugamushi. It accounts for up to23% of all febrile episodes in endemic areas of the Asia-Pacific region(5). The disease is characterized by a rise in body temperature, skinrash and severe headaches. This disease may affect the nervous system,with clinical manifestations such as delirium, stupor and musclefibrillation. The death rate varies from 1 to 60% depending on thegeographical regions. Scrub typhus, transmitted to mammals (includinghumans and cattle) through the bite of tiny trobiculid mites(arthropods) is particularly high in South-East Asia, Korea, Russia,Australia, China, Japan and India. The incidence of disease hasincreased in some countries during the past several years (6).

The causative organism is transmitted to human through the bite of tinytrobiculid mites. The organisms are found throughout the mite's body,but the highest number occurs in the salivary glands. When the mitesfeeds on mammals, including cattle, rodents or humans, the diseasecausing organisms are transmitted from the mite to the invertebrate host(subject). Srub typhus infections are usually found in people engaged inactivities that bring them inadvertently in contact with mite-infestedhabitats or any invertebrate host-carrier of these anthropods. Thesehosts may include domesticated, non-domesticated or farm animals, suchas cattle or rodents. These hosts may be carrying mites which have notbegun to feed on them. In this case, when the host is cattle, the livemites can be transferred from cattle to people. Individuals particularlysusceptiable include butchers, meatworkers, animal-farm workers andothers engaged in outdoor activities. These persons could be infected bycoming into contact with these mite-carrying animals. Additionally,rodents are capable of carrying and spreading infected mites to peoplein populated areas.

Only larval Leptotrombidium mites feed on vertebrate hosts. The larvalmites acquire O. tsutsugama through their female parent. This type ofpathogen reception is called “transovarial transmission.”

Once transmitted to the host, the organism incubates for about 10 to 12days. From 5 to 8 days after infection, a dull read rash may appear allover the body, especially on the trunk. Mortality ranges from 1 to 60%.Death either occurs as a direct result of the disease, or from secondaryeffects, such as bacterial pneumonia, encephalitis, or circulatoryfailure. If death occurs, it is usually by the end of the second week ofinfection. Despite these tragic statistics, many people around the worlddo not understand or believe how deadly scrub typhus can be until it istoo late. Unfortunately, too many people are unwittingly dancing withdeath.

FIELD OF THE INVENTION

This invention relates to detecting exposure to and identification ofmicroorganisms by the use of serodiagnostic assays, and morespecifically to detecting exposure to and identification toward atruncated protein of at least one strain of Orientia tsutsugamushi basedon its strength in reactivity. Additionally, this invention related tothe production of vaccines, passive prophylactic or therapeutic agentsand detection or identification reagents. The products produced inaccordance with this invention may be combined with otherpharmaceutically-acceptable bioactive substances.

DESCRIPTION OF PRIOR ART

Scrub typhus is caused by O. tsutsugamushi, a gram negative bacterium.In contrast to other gram negative bacteria, O. tsutsugamushi hasneither lipopoly-saccharide nor a peptidoglycan layer (1) and theultrastructure of its cell wall differs significantly from those of itsclosest relatives, the typhus and spotted fever group species in thegenus Rickettsia (33). The major surface protein antigen of O.tsutsugamushi is the variable 56 kDa protein which accounts for 10-15%of its total protein (16, 28). Most type-specific monoclonal antibodiesto Orientia react with homologues of the 56 kDa protein (16, 24, 42).Sera from most patients with scrub typhus recognize this protein,suggesting that it is a good candidate for use as a diagnostic antigen(28).

Diagnosis of scrub typhus is generally based on the clinicalpresentation and the history of a patient. However, differentiatingscrub typhus from other acute tropical febrile illnesses such asleptospirosis, murine typhus, malaria, dengue fever, and viralhemorrhagic fevers can be difficult because of the similarities in signsand symptoms. Highly sensitive polymerase chain reaction (PCR) methodshave made it possible to detect O. tsutsugamushi at the onset of illnesswhen antibody titers are not high enough to be detected (14, 19, 36).PCR amplification of the 56 kDa protein gene has been demonstrated to bea reliable diagnostic method for scrub typhus (14, 18). Furthermore,different genotypes associated with different Orientia serotypes couldbe identified by analysis of variable regions of this gene withoutisolation of the organism (14, 17, 18, 25, 39). However, geneamplification requires sophisticated instrumentation and reagentsgenerally not available in most rural medical facilities. Currentserodiagnostic assays such as the indirect immunoperoxidase (IIP) testand the indirect immunofluorescent antibody (IFA) ormicroimmunofluorescent antibody (MIF) tests require the propagation ofrickettsiae in infected yolk sacs of embryonated chicken eggs orantibiotic free cell cultures (4, 20, 30, 43).

At the present time the only commercially available dot-blot immunologicassay kits (Dip-S-Ticks) requires tissue culture grown, Renografindensity gradient purified, whole cell antigen (41). Only a fewspecialized laboratories have the ability to culture and purify O.tsutsugamushi since this requires biosafety level 3 (BL3) facilities andpractices. The availability of recombinant rickettsial protein antigenswhich can be produced and purified in large amounts and have similarsensitivity and specificity to rickettsia-derived antigens would greatlyreduce the cost, transport, and reproducibility problems presentlyassociated with diagnostic tests which require the growth andpurification of rickettsiae. Furthermore, large-scale growth andpurification of the scrub typhus rickettsiae are prohibitivelyexpensive.

Recently, a recombinant 56 kDa protein from Boryong strain fused withmaltose binding protein was shown to be suitable for diagnosis of scrubtyphus in a enzyme-linked immunosorbent assay (ELISA) and passivehemagglutination test (21, 22). Although this protein overcomes some ofthe above-described disadvantages, it still has certain inherentdisadvantages as an assay reagent because it is a fusion protein.

SUMMARY OF THE INVENTION

Accordingly, an object of this invention is a recombinant DNA constructand expressed polypeptide possessing immunogenic regions for the Karp,Kato and Gilliam strains of O. tsutsugamushi.

Another object of the invention, as described herein, is a recombinantpolypeptide encoding a portion of the 56 kDa protein of O. tsutsugamushiencoded by amino acids 80 to 456 for Karp strain SEQ ID NO.: 1, 81-453for Kato strain SEQ ID No. 4 and 81-448 for Gilliam strain SEQ ID NO.:5.

A still further object of the invention is a recombinant truncated 56kDa polypeptide which is re-folded to give a soluble moiety.

An additional object of this invention is the use of at least onerecombinant polypeptide in antibody based assays for improved methodsfor the detection of O. tsustugamushi exposure and/or identification ofat least one of its Karp, Kato or Gilliam strains in research and inclinical samples.

Yet another object of the invention is the expression of truncated r56polypeptides in different host backgrounds of bacterial strains for usein different vaccine formulations against scrub typhus infection.

These and other objects, features and advantages of the presentinvention are described in or are apparent from the following detaileddescription of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further described with reference to the drawings,in which like elements have been denoted throughout by like referencenumerals. The representation in each of the figures is diagrammatic andno attempt is made to indicate actual scales or precise ratios.Proportional relationships are shown as approximations.

FIG. 1 shows the strategy for cloning and construction of pWM1 thatexpresses the truncated recombinant 56 kDa protein antigen from O.tsutsugamushi Karp strain.

FIG. 2 shows the HPLC ion exchange profile for the purification of r56.The insert shows the Coomassie blue staining (A) and Western blotanalysis (B) of the two peak fractions at 25 (left lane) and 27 min(right lane) which contain most of the r56.

FIG. 3 shows the circular dichroism spectrum of refolded r56.

FIG. 4 shows a comparison of ELISA IgG reactivity of r56 and O.tsutsugamushi Karp strain whole cell lysate with rabbit antisera (seeTable 1).

FIG. 5 shows a scattergram of IgG ELISA reactivity of 128 Thai patientsera obtained with folded r56 and the corresponding IIP test IgG titers.

FIG. 6 shows a scattergram of IgM ELISA reactivity of 128 Thai patientsera obtained with folded r56 and the corresponding IIP test IgM titers.

FIG. 7 shows the time course of IgM and IgG reactivity of confirmedcases of scrub typhus by ELISA with folded r56 as antigen.

DETAILED DESCRIPTION

There is a critical need for rapid assays for the determination ofexposure to Orientia tsutsugamushi, the causative agent of scrub typhus.Currently available assays require bacterial antigen which must bepurified by extremely labor intensive methods after first propagatingthe organism in specialized laboratories (BSL-3). Furthermore, there iscurrently no efficacious vaccine for scrub typhus.

Recombinantly produced protein antigens of O. tsutsugamushi andrecognized by specific antibodies would greatly facilitate the practicaluse of anti-scrub typhus assays since the protein can be produced moreeconomically. Additionally, recombinant polypeptides can be used insub-unit vaccines.

In accordance with the practice of this invention, a recombinant,refolded non-fusion polypeptide expressed from a truncated r56 gene ofthe causative agent of scrub typhus, Orienta tsutsugamushi for the Karp,Kato and Gilliam strains has been produced. The invention is useful fordetecting prior exposure to scrub typhus, screening for and/oridentification of at least one infectious strain-similarity (i.e. aKarp-like, Kato-like, or Gilliam-like strain) based on its strength ofreaction toward a truncated protein and as a component in vaccineformulations and production of immune globulins for passive prophylaxisand immunity in subjects.

The 56 kDa protein of O. tsutsugamushi is extremely abundant in thebacteria and is highly immunogenic. Although the use of recombinant 56kDa protein from O. tsutsugamushi has been reported, it was produced asa fusion peptide which creates a number of inherent disadvantages,including reduced immunogenicity due to improper folding of the bacteriapolypeptide. To overcome these problems a non-fusion, recombinantpolypeptide from 56 kDa protein was produced using the followingalternative procedures designated herein as PROCEDURES I and II toexpress and purify r56 from the Karp, Kato and/or Gilliam strains.Furthermore, as illustrated herein for the production of SEQ ID No. 1,in order to ensure proper folding of the polypeptide after translation,and therefore enhanced immune recognition, a truncated recombinant 56kDa gene was created with the truncation created at specific points (SeqID No. 1). The truncated 56 kDa gene is then expressed using efficientexpression systems. This truncated, recombinant polypeptide is then useas antigen in antibody based assays and to induce an immune responseagainst scrub typhus. The specification generally uses the Karp strainfor illustrative purposes only, as the following examples apply to otherstrains of O. tsutsugamushi, including the Kato and Gilliam strains.

EXAMPLE 1

Cloning and Expression of Recombinant 56 kDa Gene.

As shown in FIG. 1, a primer pair (56F(226/261),5′-TTGGCTGCACATATGACAATCGCTCCAGGAT TTAGA-3′ (Seq. ID No. 2) and56R(1409/1363), 5′-CTTTCTAGAAGTATAAGCTAACCCGGATCC AACACCAGCCTATATTGA-3′(Seq. ID No. 3) was designed using the nucleotide sequence of the openreading frame for the Karp 56 kDa protein (34). The respectiverestriction sites for Nde I and BamH I are underlined and the newinitiation codon and reverse complement of the new stop codon are shownin bold and italic, respectively. The forward primer 56F(226/261)contained the methionine initiation codon, at residue 80, which is partof the Nde I recognition sequence. The reverse primer 56R(1409/1363)created an alteration of the tyrosine codon at residue 457 to a stopcodon and contained a BamH I site. The coding sequence from amino acid80 to 456 was amplified by polymerase chain reaction (PCR), using theabove primers, from DNA isolated from plaque-purified O. tsutsugamushiKarp strain grown in irradiated L929 cells (18). The truncated 56 kDagene was amplified in a mixture of 400 mM each of deoxynucleotidetriphosphate, 1 mM of each primer, 1.5 U of Taq polymerase(Perkin-Elmer, CA) in 10 mM Tris-HCl buffer, pH 8.3, 1.5 mM MgCl², and50 mM KCl. The PCR reaction was started with 15 sec at 80° C., 4 min at94° C., and followed by 30 cycles of 94° C. for 1 min, 57° C. for 2 minand 72° C. for 2 min. The last cycle was extended for 7 min at 72° C.The amplified fragment (1.18 kb) was digested with Nde I (BioLab, MA)and BamH I (Life Technology, MD) and ligated with doubly digestedexpression vector. Any plasmid or viral expression system can be used aslong as polypeptide is expressed. The preferred expression system is theplasmid system pET11a (Novagen, WI) (FIG. 1) to yield the expressionsystem pWM1. The E. coli strain HB101 was transformed with the ligationmixture and colonies screened for inserts with the right size andorientation.

Expressed r56 is constructed such that the N-terminal 79 residues or theC-terminal 77 residues of the intact 56 kDa protein, as deduced from theopen reading frame of its encoding gene, is not present. Both regionsdeleted were predicted to be relatively hydrophobic and be responsiblefor association with the rickettsial outer membrane. Truncation of thesetermini facilitate the refolding of the expressed polypeptide and favorsits solubility in aqueous solutions and simplification of handling.

Purification of the 56 kDa Protein.

Plasmids carrying the insert of the truncated and amplified 56 kDa geneare transformed into the expression host E. coli BL21. The optimum timeand IPTG concentration for r56 expression is determined. Recombinant E.coli expressing r56 are grown overnight at 37° C. with shaking. Cellpellets from 100 ml cultures are resuspended in 3 ml of buffer A (20 mMTris-HCl, pH 8.0), containing 5 mM EDTA and 1 mM PMSF. Ultrasonicdisruption of the cell is performed with cooling on ice. Disrupted cellextract is centrifuged at 8,000×g for 30 min. The pellets are vortexedto a homogeneous suspension with 2 M urea in buffer A, placed on ashaker at room temperature for an additional 10 min, centrifuged for 5min at 14,000 rpm in an Eppendorf centrifuge (model 5415). The entireprocess is then repeated with 4 M urea in buffer A. Finally the pelletsare dissolved in 8 M urea in buffer A and applied onto an HPLC ionexchange (DEAE) column (Waters, 0.75 cm×7.5 cm) for fractionation.Proteins are eluted with a linear gradient of buffer B and buffer C (6 Murea and 2 M NaCl in buffer A) from 0.0 to 0.4 M NaCl over 30 min at aflow rate of 0.5 ml/min. Fractions are collected, typically at one minper fraction. For a typical run, approximately 200 μl of extractobtained from a total of 10 ml culture is loaded onto the column (FIG.2). The presence of r56 in fractions was detected by dot-blotimmunoassay. Positive fractions with significant amounts of protein,presumably containing expressions of the truncated and amplified 56 kDagene, are also analyzed by SDS-PAGE and Western blotting.

Testing for Polypeptide Expression by Dot-Blot Immunoassay.

Fractions collected from HPLC are screened for r56 polypeptide bydot-blot assay. A 2 μl sample of each eluted fraction is diluted into200 μl of water and applied to a well of a 96-well dotblotter(Schleicher and Schuell). After drying under vacuum for 5 min, thenitrocellulose membrane is blocked with 5% nonfat milk for 30 min, thenincubated with monoclonal antibody Kp56c specific for Karp 56 kDaprotein antigen (23) for one hr, washed 4 times with phosphate buffersaline (PBS) 5 min each time, and incubated with peroxidase conjugatedgoat anti-mouse IgG (H+L) (Bio-Rad Laboratories) for 30 min. Afterwashing with PBS 5 times for 5 min, substrate solution containing 5:5:1ratio of TMB peroxidase substrate, hydrogen peroxide solution, and TMBmembrane enhancer (Kirkegaard and Perry Laboratories) is added onto thenitrocellulose membrane. The enzymatic reaction is stopped after 2 minby washing the membrane in distilled water. The above-described test canbe incorporated into any dot-blot, spot or dipstick type test structure.These structures are extensively described in the prior art.

Confirmation of Polypeptide Identity.

Confirmation of the identity of the polypeptide is confirmed by aminoacid sequence analysis of SDS-PAGE purified, CNBr cleaved fragments ofthe peak fractions (7). The sequences are identical to that deduced fromnucleotide.

Refolding of r56.

HPLC fractions, in 6M urea, containing peak r56 polypeptide are pooledand sequentially dialyzed against 4 M urea and 2 M urea in buffer A andfinally with buffer A only. The final dialysis is against buffer A withtwo initial changes of buffer for 30 min each, and finally overnight at4° C. r56 is properly folded since the polypeptide remains soluble inbuffer A with no urea present.

Circular Dichroism (CD) Spectrum of r56.

The circular dichroism spectrum of refolded r56 was measured on a JASCOmodel 715 in Dr. Ettore Apella=s laboratory in NIH, Bethesda, Md. Datawere analyzed by Dr. Latchezar I. Tsonev, Henry Jackson Foundation,Rockville, Md., at a protein concentration of 117 μg/ml in 20 mM TrisHCl, pH 8.0 and the calculated molecular weight of 40,903 dalton.

The CD spectrum of the refolded polypeptide shows that the secondarystructure is approximately 38% α-helical, 13% β-sheet and 50% randomcoil (15) (FIG. 3). This experimental data is similar to that predictedby correctly folded, truncated 56 kDa protein, based on amino acidsequence from nucleic acid sequence (34).

EXAMPLE 2

Use of r56 Polypeptide in Antibody Based Identification Assays.

ELISA Assay Method

The microtiter plates are coated with antigens diluted in PBS overnightat 4° C. and blocked with 0.5% boiled casein for 1 hr, rinsed with PBStwice, 5 min each time. Patient sera are diluted 1:400 with 20 μg/ml ofcontrol protein extracts purified from E. coli BL21 using a procedureidentical to that used for purifying r56 (fractions 21-32 pooled fromgradients equivalent to FIG. 2), pre-absorbed for about 1 hr at roomtemperature, and then added to the ELISA plates. The plates areincubated for 1 hr at room temperature, washed four times with 0.1%Triton X-100 in PBS. Peroxidase conjugated mouse anti-human IgG (Fcspecific) (Accurate) diluted 1:8000 and goat anti human IgM (μ chainspecific) (Kirkegaard & Perry) are then added. After 1 hr incubation atroom temperature, the plates are washed four times with 0.1% TritonX-100 in PBS and the last wash is with PBS only before the addition ofsubstrate ABTS (Kirkegaard & Perry). The ODs at 405 nm are read after 15min incubation at room temperature. Rabbit sera were diluted 1:250 withPBS only. All procedures are the same as for detection of humanantibodies except that rabbit sera is not preabsorbed with proteinpreparations from BL21 and peroxidase conjugated goat anti-rabbit IgG(Kirkegaard & Perry) diluted 1:2,000 is used.

The recombinant r56 polypeptide contains only a portion of the 56 kDaprotein, the major antigen that is used to differentiate antigenic typesof Orientia. In addition rickettsial whole cell lysate contains numerousother protein antigens besides intact 56 kDa antigen. A comparison ofELISA IgG reactivity of r56 and O. tsutsugamushi Karp strain whole celllysate with rabbit antisera is shown (FIG. 4). The dotted linesrepresent the mean+2 standard deviations of reactivity of the normalrabbit sera. The solid line is the linear regression of the data for the22 anti-Orientia rabbit sera tested (r=0.81). Eight control normalrabbit sera (open diamonds); five antisera against non-rickettsialantigens (open triangles): eight antisera to Rickettsiales other thanOrientia (open squares); and 22 antisera to eight antigen prototypes ofO. tsutsugamushi (solid circles) are compared. Positive breakpoints(mean+2SD) for reactivity of both r56 and whole cell Orientia lysate(WCEX) and standard ELISA using eight normal rabbit (ODs of 0.27 and0.38), respectively, are established. (FIG. 4, Table 1). None of theeight rabbits immunized with other species of Rickettsiales or the fiveantisera prepared against either L-cell, yolk sac, or E. coli exhibitreactivity higher than the cutoff for WCEX while one rabbit antiserumagainst primary chick embryo reacted barely above the breakpoint withr56 (OD of 0.28) (FIG. 4, Table 1). On the other hand 20 of 22 rabbitantisera against the eight Orientia antigenic prototypes react slightlyabove the breakpoint with r56 and all sera exhibit positive ELISA withWCex (FIG. 4, Table 1). Although the r56 antigen exhibits lower ELISAreactivity at the amount employed than that obtained with WCex, theOrientia rabbit antisera exhibit a very good correlation of ELISAreactions to the two antigens (r=0.8, n=22). One Kato antiserum and oneTA686 antiserum which exhibit relatively low positive ELISA reactivitywith WCex does not react, significantly, with r56 antigen (Table 1).Consequently, the ELISA with folded r56 gives equivalent results as thestandard ELISA in the detection of Orientia-specific antibodies by ELISA(specificity-92.3%, sensitivity-90.9%, accuracy-91.4%) with WCEx ELISAas the reference assay) even though r56 is only a truncated portion ofone of the complex antigens found in WCex.

TABLE 1 Comparison of ELISA reactivity of purified Karp whole celllysate and folded r56 with rabbit antisera. Antisera against ELISAODs(405 nm) of whole cell lysate different antigens (corresponding r56result) O. tsutsugamushi strain Karp 0.94 (0.58), 1.87 (1.04), 1.81(0.80), 1.83 (0.81) Kato 0.46 (0.22), 1.02 (0.50), 1.16 (0.77), 1.27(0.58) Gilliam 0.54 (0.42), 1.20 (0.54) TH1817 1.67 (0.59), 1.12 (0.60),1.29 (0.53), 0.83 (0.47) TA678 0.59 (0.48) TA686 0.71 (0.26), 1.52(0.86) TA716 1.24 (0.48), 1.14 (0.51) TA763 1.79 (0.72), 1.57 (0.89),1.18 (0.82) Other Rickettsiales R. prowazekii 0.08 (0.12) R. typhi 0.18(0.08) R. rickettsii 0.06 (0.04), 0.15 (0.14) R. conorii 0.10 (0.11),0.07 (0.11) E. sennetsu 0.01 (0.05) E. risticii 0.01 (−0.01) Nonrickettsial antigens Yolk sac 0.22 (0.08) L929-cell 0.01 (−0.08) Primarychick 0.20 (0.28) embryo RAW 264.7 cells 0.22 (0.14) E. coli HB101 0.32(0.11) No antigen 0.135 + 0.123 (0.093 + 0.088) control (n = 8) ^(a)ODvalues listed are the difference between data with antigen and withoutantigen.Comparison of r56 ELISA with IIP Test with Human Sera.

Seventy-four sera from healthy Thai soldiers were used to establish anELISA break point for positive reactions (mean+2 SD) with r56 asantigen. These are 0.05+0.06=0.11 OD for IgG, and 0.032+0.032=0.064 ODfor IgM at 1:400 serum dilution. The r56 ELISA ODs of 128 sera frompatients suspected of scrub typhus from Korat, Thailand were comparedwith the IgG and IgM titers determined by an IIP method using a mixtureof intact Karp, Kato, and Gilliam prototypes of Orientia. The IIP methodused was described previously (20, 38) (FIGS. 5 and 6). Using IIP titersas the gold standard, the sensitivity, specificity, and accuracy valuesof ELISA results with the 128 test sera are calculated using differentpositive breakpoints for the IIP test (Table 2).

TABLE 2 Comparison of efficiency of r56 ELISA with the indirectimmunoperoxidase assay (IIP) for 128 Thai patient sera. No. pos. ELISAsera by % % % Titer Ig IIP Sensitivity Specificity Accuracy 1:50  IgG 6882% 92% 87% IgM 56 91% 92% 91% 1:200 IgG 61 92% 93% 92% IgM 52 98% 92%95% 1:400 IgG 57 90% 93% 95% IgM 47 100%  93% 93%

Sera from 13 isolate and PCR-confirmed cases of scrub typhus wereanalyzed to characterize the kinetics and magnitude of the IgM and IgGimmune responses as measured by IIP test titers and by r56 ELISA ODs.Representative data are shown in FIG. 7 and Table 3. Four sera from 4different cases were available from the first week after onset of fever(days 4-7). All are positive by IIP for both IgM and IgG with titersbetween 3200 and 12,800 for all cases. In contrast, by ELISA, KR5 (day4, Table 3) has very low IgM and IgG ODs and KR20 is negative for IgMeven at day 7 while the other two sera (KR8, KR25) are more reactive byIgM assay than IgG. Sixteen sera from 12 cases were collected 8-14 dayspost onset of fever. By IIP both IgM and IgG titers are again high andwithin one two-fold dilution for all of these sera except the day 10serum from KR23 which also has the lowest IgM and IgG ELISA OD's (Table3, FIG. 7). Except for three other sera from days 8-10 (KR5, KR43, KR51)which also had low IgM ODs, most sera has similar IgG and IgM ELISAreactions. Five sera from four cases were obtained in weeks 3-4 afterinfection. Two of the cases (KR8, KR20) exhibit a decrease in IgM ODs byELISA at this time point which are not apparent by IIP assay while theother reactions all remain strong. In weeks 5-6 after infection two of 5sera from different patients decline in IIP IgM titers (but not IgGtiters) while three sera decline significantly in ELISA IgM and one byELISA IgG. In striking contrast, KR27 maintain high levels of specificantibody as measured by all assays from 10 to 39 days (Table 3). Withall six sera collected from six different cases 95-202 days post onsetof illness, IgM IIP titers and both IgM and IgG ELISA ODs dropsignificantly; in contrast, only one of the sera exhibit a decline inIgG IIP titers (FIG. 7).

TABLE 3 Comparison of IIP test titers with ELISA r56 OD's obtained withhuman sera from confirmed cases of scrub typhus. Days post IIP TestTiter r56 ELISA (OD) Patient Onset of fever IgM IgG IgM IgG KR5 4 3,2003,200 0.10 0.31 KR5 10 6,400 12,800 0.34 1.26 KR5 29 1,600 12,800 0.070.63 KR8 5 12,800 12,800 1.55 1.18 KR8 10 6,400 6,400 1.48 0.92 KR8 2612,800 12,800 0.71 0.85 KR8 47 12,800 12,800 0.57 0.90 KR8 137 50 3,2000.05 0.35 KR10 10 12,800 6,400 1.30 1.15 KR10 201 200 6,400 0.053 0.20KR20 7 3,200 6,400 0.01 1.00 KR20 22 3,200 6,400 0.44 0.82 KR20 27 6,40012,800 0.24 0.50 KR20 95 200 6,400 0.03 0.13 KR23 10 200 800 0.14 0.32KR23 14 1,600 3,200 0.97 1.50 KR23 29 800 3,200 0.26 1.32 KR25 7 12,80012,800 1.34 0.84 KR25 11 6,400 6,400 1.54 0.86 KR27 10 3,200 6,400 1.301.10 KR27 12 6,400 12,800 1.30 1.20 KR27 24 3,200 12,800 1.14 1.23 KR2739 3,200 12,800 1.03 1.20 KR43 9 6,400 6,400 0.27 0.85 KR43 12 6,4006,400 0.96 1.17 KR43 13 12,800 12,800 1.16 0.93 KR51 8 3,200 12,800 0.390.74 KRS1 11 6,400 6,400 1.04 1.32

The excellent sensitivity and specificity of the r56 ELISA in comparisonwith those of the IIP assay suggest that one protein antigen, i.e.truncated r56, is sufficient for detecting anti-Orientia antibody insera from patients with scrub typhus. Use of a single moiety inrecombinant form improves efficiency of the assay and will reduce costper assay, significantly.

EXAMPLE 4

Induction of Protective Immune Response.

Because of the significant antibody response exhibited after exposurewith O. tsutsugamushi in rabbits and humans, and the excellentrecognition pattern of r56 polypeptide compared to whole cell extracts,the r56 polypeptide is a good candidate vaccine component.

Two strains of either relatively outbred mice (CD1) or an inbred strain(C3H) were immunized, with adjuvant with the r56 polypeptide. At varioustimes after administration of the polypeptide the animals werechallenged with live O. tsutsugamushi.

The protective efficacy of administration of r56 polypeptide is shown intable 4.

TABLE 4 Protection of Mice by Immunization with r56 Challenge Strain ofDose/Mouse date post % Experiment mice (adjuvant) immunizationProtection I C3H 25 μg 3 weeks 100%  (incomp. Freunds) II CD1 25 μg 4months 60% (Titer Max) III CD1 2 μg 4 weeks 60% (Titer Max)

Karp, Kato and Gilliam Strains

The variable 56 kDa major outer membrane protein of Orientiatsutsugamushi is the immunodominant antigen in human scrub typhusinfections. The gene encoding this protein from Gilliam strain andKatostrain was cloned into the expression vector pET24a. The recombinantprotein (r56) was expressed as a truncated non-fusion protein (aminoacid 81 to amino acid 488 of the open reading frame for Gilliam andamino acid 81 to amino acid 453 of the open reading frame for Katostrain). Both protein formed an inclusion body when expressed inEscherichia coli BL21. The refolded r56 (Gilliam) and r56(Kato) werereactive to sera from scrub typhus patient. Three recombinant antigens,r56(karp), r56 (Gilliam), and r56(Kato) were mixed at an equal ratio andused as the antigen in an ELISA. A panel of patient sera exhibiting awide range of reactivity was employed to compare the reactivity of mixedrecombinant r56 antigens with mixed whole cell antigens. The ELISAresults correlated well to those obtained using whole cell lysate fromthe corresponding strains as the coating antigen in the ELISA. Theseresults strongly support that the mixture of the recombinant proteinshas the potential to be used as a diagnostic reagent, exhibiting broadsensitivity and high specificity for scrub typhus infection and inproduction of immune globulins, vaccines, and therapeutic agents. Therecombinant r56(Gilliam) and r56(Kato) have the potential to replace thedensity gradient-purified, rickettsia-derived, whole cell antigencurrently used in the commercial dip-stick assay available in the USA.

The molecular cloning, expression, purification, and refolding of thetruncated non-fusion 56 kDa protein from Gilliam strain, r56(Gilliam),and from Kato strain, r56(Kato) will now be described. The refoldedr56(Gilliam) reacted strongly with monoclonal antibody (mAb) RK-G3C51but did not react with mAb E+95. The r56 (Kato) reacted with E+95, butnot with RK-G3C51. The strain variations of Orientia are welldocumented. In order to develop a diagnostic reagent that will detectmost cases of scrub typhus infection, different serotype antigens needto be included in the antigen cocktail employed. A mixture of threepurified recombinant r56 (Karp, Gilliam and Kato) was evaluated for itsreactivity with 20 patient sera which exhibited wide range of reactivitywith whole cell lysate cocktail of strains Karp, Gilliam, and Kato in astandard ELISA for diagnosis of scrub typhus. The ELISA results of usingmixture of r56 correlated well to those obtained using the mixture ofcorresponding strains of whole cell lysate. These results stronglysuggest that the recombinant proteins have the potential to be used asdiagnostic reagents, exhibiting broad sensitivity and high specificityfor scrub typhus infection.

Bacterial Strains and Vectors. Escherichia coli HB101 was used forcloning and E. coli BL21(DE3) was used for overexpression of proteinsunder the control of phage T7lac promoter (26). The plasmid vector usedwas pET-24a (Novagen, Madison, Wis.). Plaque-purified O. tsutsugamushiGilliam and Kato strains were grown in irradiated L929 cells was usedfor preparation of the genomic DNA (11).

Cloning of the gene for the r56 (Gilliam) into the expression vectorpET24a. A primer pair 56FGm(784/819), 5′ T T A G C T G C G C.dwnarw.A TA T G A C A A T T G C A C C A G G A T T T A G A 3′ (SEQ ID NO. 6) andr56RGm (1929/1894) 5′ A T G A G C T A A C C C G.dwnarw.G A T C C A A C AC C A G C C T A T A T T G A 3′ (SEQ ID NO. 7) was designed using thenucleotide sequence of the open reading frame for the Gilliam 56 kDaprotein (27). The respective restriction sites for Nde I and BamH I areunderlined and bold. The forward primer 56FGm(784/819) contained themethionine initiation codon, at residue 81, which is part of the Nde Irecognition sequence. The reverse primer 56RGm(11929/1894), mutated thetyrosine codon at residue 448 to a stop codon and contained a BamH Isite. The coding sequence from amino acid 81 to 448 was amplified by PCRfrom DNA isolated from O. tsutsugamushi Gilliam strain. Cloning of thegene for the r56 (Kato) into the expression vector pET24a. A primer pair56FKt(785/820), 5′ T T A G C T G C A C.dwnarw.A T A T G A C A A T C G CG C C A G G A T T T A G A 3′ SEQ ID NO. 8 and r56RKt (1945/1910), 5′ A TA A G C T A A C C C G.dwnarw.G A T C C A A G A C C A G C C T A T A T T GA 3′ (SEQ ID NO. 9 was designed using the nucleotide sequence of theopen reading frame for the Kato 56 kDa protein (31). The respectiverestriction sites for Nde I and BamH I are underlined and bold. Theforward primer 56FGm(784/819) contained the methionine initiation codon,at residue 81, which is part of the Nde I recognition sequence. Thereverse primer 56RGm (11929/1894), mutated the tyrosine codon at residue448 to a stop codon and contained a BamH I site. The coding sequencefrom amino acid 81 to 448 was amplified by PCR from DNA isolated from O.tsutsugamushi Gilliam strain.

Cloning of the gene for the r56 (Kato) into the expression vectorpET24a. A primer pair 56FKt(785/820), 5′ T T A G C T G C A C↓A T A T G AC A A T C G C G C C A G G A T T T A G A 3′ and r56RKt (1945/1910), 5′ AT A A G C T A A C C C G ↓ G A T C C A A G A C C A G C C T A T A T T G A3′ was designed using the nucleotide sequence of the open reading framefor the Kato 56 kDa protein (31). The respective restriction sites forNde I and BamH I are underlined and bold. The forward primer56FGm(784/819) contained the methionine initiation codon, at residue 81,which is part of the Nde I recognition sequence. The reverse primer56RGm (11929/1894), mutated the tyrosine codon at residue 448 to a stopcodon and contained a BamH I site. The coding sequence from amino acid81 to 448 was amplified by PCR from DNA isolated from O. tsutsugamushiGilliam strain.

The two truncated 56 kDa genes were amplified in a mixture of 400 mMeach of deoxynucleotide triphosphate, 1 mM of each primer, 1.5 U of Taqpolymerase (Perkin-Elmer-Cetus, Norwalk, Conn.) in 10 mM Tris-HClbuffer, pH 8.3, 1.5 mM MgCl₂, and 50 mM KCl. The PCR reaction wasstarted with 15 sec at 80° C., 4 min at 94° C., and followed by 30cycles of 94° C. for 1 min, 57° C. for 2 min and 72° C. for 2 min. Thelast cycle was extended for 7 min at 72° C. The amplified fragments wasdigested with Nde I (New England BioLabs, Beverly, Mass.) and BamH I(GIBCO-BRL Life Technology, Gaithersburg, Md.) and ligated with doublydigested expression vector pET24a. E. coli HB101 was transformed withthe ligation mixture and colonies screened for inserts with the rightsize and orientation.

Procedure I

Expression and purification of the r56 (Gilliam) and r56(Kato). Plasmidscarrying the insert were transformed into the expression host E. coliBL21. The optimum time and isopropyl-□-D-thiogalactopyranoside (IPTG)concentration for inducing r56 expression was determined. Recombinant E.coli expressing r56 (Gilliam) were propagated overnight in 2X YT (16 gbacto-tryptone, 10 g bacto-yeast extract, and 5 g NaCl per liter ofdistilled water, pH 7.0) at 37° C. with shaking. Cell pellets from 100ml cultures were resuspended in 3 ml of buffer A (20 mM Tris-HCl, pH8.0), containing 5 mM EDTA. Ultrasonic disruption of the cell wasperformed using setting 3 on a Sonicator Ultrasonic Liquid ProcessorModel XL2020 with standard tapered microtip (Heat Systems, Inc.,Farmingdale, N.Y.), six times for 20 sec with cooling on ice for 1 minbetween each sonication. Disrupted cell extract was centrifuged at8,000×g for 30 min. The pellets were vortexed to a homogeneoussuspension with 2 M urea in buffer A, placed on a shaker at roomtemperature for an additional 10 min, centrifuged for 5 min at 14,000rpm in an Eppendorf centrifuge (model 5415). The entire process was thenrepeated with 2% sodium deoxycholate in buffer A. Finally the pelletswere dissolved in 8 M urea in buffer A. The supernant was applied ontoan high pressure liquid chromatography (HPLC) ion exchange (DEAE 5PW)column (Waters Associates, Milford, Mass.) (0.75 cm×7.5 cm) forfractionation. Proteins were eluted with a linear gradient of buffer B(6 M urea in buffer A) and buffer C (6 M urea and 2 M NaCl in buffer A)from 0.0 to 0.4 M NaCl over 30 min at a flow rate of 0.5 ml/min.Fractions were collected at one min per fraction. The presence of r56 infractions was detected by dot blot immunoassay. Positive fractions withsignificant amounts of protein were analyzed by SDS-PAGE and Westernblotting.

Dot blot immunoassay. A 2 μl sample of each eluted fraction was dilutedinto 200 μl of water and applied to a well of a 96-well dotblotter(Schleicher and Schuell, Keene, N.H.). After drying under vacuum for 5min, the nitrocellulose membrane was blocked with 5% nonfat milk for 30min, then incubated with antibody specific for Gilliam or Kato 56 kDaprotein antigen for 1 hr, washed 4 times with phosphate buffer saline(PBS) 5 min each time, and incubated with peroxidase conjugated goatanti-mouse IgG (H+L) (Bio-Rad Laboratories, Richmond, Calif.) for 30min. After washing with PBS 5 times for 5 min, substrate solutioncontaining 5:5:1 ratio of TMB (tetramethylbenzidine) peroxidasesubstrate, hydrogen peroxide solution, and TMB membrane enhancer(Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was added ontothe nitrocellulose membrane. The enzymatic reaction was stopped after 2min by washing the membrane in distilled water.

SDS-PAGE and Western Blot analysis. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis wasperformed with the mini-protein II Dual Slab Cell System (8.2 cm×7.2cm×0.75 cm, Bio-Rad). The stacking gel and separation gel contained 4%and 10% acrylamide (acrylamide:bisacrylamide ratio was 30:1),respectively. Electrophoresis was carried out at constant voltage of 125V for 75 min. The gels were either stained with Coomassie Blue R orelectroblotted onto nitrocellulose membrane. Immunodetection of theWestern blot was the same as described for the dot blot immunoassay.

Refolding of r56. Refolding of r56(Gilliam) and r56(Kato) in 6 M urea inbuffer A were achieved by sequential dialysis with 4 M urea and 2 M ureain buffer A and finally with buffer A only. The peak fractions from theDEAE column were combined and dialyzed against 8 volumes of 4 M urea inbuffer A for 30 min at room temperature followed with one change of thedialysis solution and dialyzed for an additional 30 min. The sameprocedure was repeated with 2 M urea in buffer A. The final dialysis wasagainst buffer A with two initial changes of buffer for 30 min each, andfinally overnight at 4° C.

Human sera. Patient sera were collected from Pescadore Islands in 1976(2).

ELISA. 96 well microtiter plates were coated overnight at 4° C. withantigens diluted in PBS and blocked with 0.5% boiled casein for 1 hr,rinsed with PBS twice, 5 min each time. Linbro U plates (Cat. No. U76-311-05, ICN, Costa Mesa, Calif.) were used for assays with rabbitsera while Microtest III tissue culture plates (Falcon #3072) wereemployed with human sera. Patient sera were diluted 1:100 in PBS. Theplates were incubated for 1 hr at room temperature, washed four timeswith 0.1% Triton X-100 in PBS. Peroxidase conjugated mouse anti-humanIgG (Fc specific) (Accurate Chemical and Scientific Corp., Westbury,N.Y.) diluted 1:2000. After 1 hr incubation at room temperature, theplates were washed four times with 0.1% Triton X-100 in PBS and the lastwash was with PBS only before the addition of substrate ABTS (Kirkegaard& Perry). Optical densities (ODs) at 405 nm were measured at 10 min and15 min at room temperature.

Table 5 lists the ELISA data of 20 patient sera. The ELISA results usingthe mixture of three recombinant r56 polypeptides correlated well tothose obtained using whole cell lysate from the corresponding strains asthe coating antigen. A basic problem in the design of diagnostic testsfor Orientia is that numerous serotypes exist. Eight prototypes(Gilliam, Karp, Kato, TA686, TA716, TA678, TA763, TH1817) have beenwidely used as reference strains for MIF serotyping of isolatescollected throughout the areas endemic for Orientia (7, 24). In recentyears several additional serotypes from Japan and Korea have beenrecognized (5, 22, 33). We have recently characterized more than 200Orientia isolates by restriction fragment length polymorphism (RFLP)analysis of four different antigen gene homologues following theiramplification by polymerase chain reaction (6, 11). 45 RFLP varianttypes were identified. The dominant human immune response is against thevariable 56 kDa outer membrane protein which is the major antigendistinguished in serotyping. Some of the antigenic serotypes found inJapan and Taiwan have recently been further subdivided by RFLP analysisof their 56 kDa genes (10, 18, 29). Both specific and cross-reactivedomains exist in different homologues of this protein. DNA sequenceanalysis of 56 kDa genes from various serotypes has revealed that thesequences may be divided into four conserved and four variable domains(19). These conserved domains of 56 kDa protein may account for thecross-reactivity of antisera against diverse serotypes while thevariable domains are very likely responsible for some of the serotypespecificity observed in Orientia. The r56 recombinant proteins lack mostof the conserved regions of the 56 kDa protein at both the N- andC-terminus. The conserved regions between the first and the secondvariable domain and between the second and the third variable domain arerelatively short. Consequently, the broad reactivity of r56 may be dueto the conserved region located between the third and the fourthvariable domain which is about 160 residue long. The four variabledomains are responsible for the strain specificity in serological tests.The O. tsutsugamushi strains Karp, Gilliam, and Kato have been shown tobe antigenically distinct. They were isolated from different geographicareas (Karp from New Guinea, Gilliam from Burma, Kato from Japan).Recently a rapid flow assay for diagnosis of scrub typhus using r56(Karp) (36, 37) was developed. To improve upon the broad reactivity ofthis RFA, r56 antigens were produced from strains Gilliam and Kato to beincluded in the RFA for future evaluation at clinical sites.

In summary, the 56 kDa major variable outer membrane protein antigen ofO. tsutsugamushi is the immunodominant antigen in human infections.Further, the strain variations of Orientia are well documented. In orderto develop a diagnostic reagent that will detect most cases of scrubtyphus infection, the preferred embodiment of the invention includes ther56 Karp antigen alone, when prepared by PROCEDURE II or in combinationwith or b. most preferably, a combination of different serotype antigensin the antigen cocktail employed.

The gene encoding this protein from the Karp strain (amino acid 80-456,designated as r56) was cloned, expressed, and purified in accordancewith PROCEDURE I. In following PROCEDURE I relative to the Kato andGilliam strains, the 56 kDa protein from the Kato strain and the Gilliamstrain were expressed with slight modifications to the procedure(PROCEDURE I) that was used to express and purify r56 from the Karpstrain. This modification is attributable to the use of different primerin the production of each of the r56 Karp (SEQ ID NO.1), r56 Kato (SEQID NO.4), and r56 Gilliam (SEQ ID NO.5) polypeptides. The r56 Gilliamand r56 Kato are truncated at both the N and C-termini, and exhibitedthe expected size by SDS-PAGE (amino acids 81-448 for r56 Gilliam, totalof 368 amino acids; amino acids 81-453 for r56 Kato, total of 373 aminoacid). The r56 Gilliam did not react with monoclonal antibody E+95 butreacted strongly with RK-G3C51. The r56 Kato reacted with E+95, but notwith RK-G3C51. These three r56 antigens were mixed at an equal ratio andused as the antigen in an ELISA. A panel of patient sera exhibiting awide range of reactivity was employed to compare the reactivity of mixedrecombinant r56 antigens with mixed whole cell antigens. The ELISAresults correlated well to those obtained using whole cell lysate fromthe corresponding strains as the coating antigen in the ELISA. Theseresults provide strong scientific evidence which supports that themixture of the recombinant proteins has the potential to be used as adiagnostic reagent, exhibiting broad sensitivity and high specificityfor scrub typhus infection.

Similarly, inventor had further developed as a further embodiment ofthis invention, an improved method (PROCEDURE II) for the production ofKarp r56, Kato r56 and Gilliam r56. Surprisingly, the final productsprepared in accordance with this new method were produced insubstantially higher concentration and purity and with less impuritiesand less aggregates as compared to the products prepared by the previousprocess (PROCEDURE I) disclosed herein. More specifically, the improvedmethod (PROCEDURE II) is as follows:

Procedure II

1. Expression of r56: Plasmids carrying the insert were transformed intothe expression host E. coli BL21. Recombinant E. coli expressing r56were induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) in thelog phase and propagated in LB medium over night at 37° C. with shaking.

2. Purification of r56 polypeptide: The r56 polypeptides were expressedas inclusion bodies (IB) in E. coli BL21. Cell pellets were re-suspendedin buffer A (20 mM Tris-HCl, pH 8.0), containing 5 mM EDTA and 0.1 mM ofphenylmethylsulfonyl fluoride (PMSF). The cells were disrupted bypassing through microfluidizer three times and the cell extract wascentrifuged at 8,000×g for 30 min. The pellets were extracted with 2 Murea in buffer A and dissolved in 8 M urea containing 10-20 mMDTT for2.5 to 5 mg/ml of r56. After incubation at room temperature for at least20 minutes, the sample solution was centrifuged at 8,000×g for 5minutes. The clear supernatant (<1/10 of the column volume) was appliedto size-exclusion columns TSK P3000SW (21.5 mm×50 cm) -tandem TSKP4000SW (21.5 mm×100 cm) column equilibrated with 8 M urea and 1 mM DTTin 20 mM Tris-HCl, pH 7.8 (buffer B). Peak fractions containing the r 56polypeptide were pooled and loaded into the anion-exchange DEAE column(21.5 mm×30 cm). The bound r56 was eluted with a linear gradient of NaClfrom 0 to 0.4 M in buffer B over 30-60 min at a flow rate of 5 ml/min.

3. Refolding of the purified r56 polypeptide: Refolding of r56 in bufferB were achieved by sequential dialysis with 6 M urea, 4 M urea, and 2 Murea in buffer A and finally with buffer A only. The peak fractions fromthe DEAE column were combined and dialyzed against 8 volumes of 6 M ureain buffer A for 30 minutes at 4 degrees Celsius (4 C) followed with twochanges of the dialysis solution and dialyzed for a total of anadditional 60 minutes. The same procedure was repeated with 4 M and 2 Murea in buffer A, except 0.3 uM of oxidized form of glutathione wasincluded in the 4M urea solution. The final dialysis was against bufferA with two initial changes of buffer for 30 min each, and finallyovernight at 4 C.

Protection Efficacy data for (a) r56 (Karp only), (b) r56 (Kato only)and (c) a mixture of r56 Karp, r56 Kato and r56 Gilliam, challenged byKato.

TABLE 5 Immunoprotection of Swiss Outbred CD1 Mice from Orientiatsutsugamushi Karp Strain with Kp r56 Vaccine using Freund's IncompleteAdjuvant and Alum + CpG. VACCINE ADJUVANT BOOST PROTECTION SEROLOGY PBSFIA — 38.5% 0.10 ± 0.25 PBS FIA Boost 52.9% 0.06 ± 0.06 PBS Alum-CpG —41.2% 0.12 ± 0.09 PBS Alum-CpG Boost 30.8% 0.06 ± 0.07 Kp r56 FIA — 100% 1.56 ± 0.15 Kp r56 FIA Boost   95% 1.49 ± 0.37 Kp r56 Alum-CpG —76.9% 1.48 ± 0.08 Kp r56 Alum-CpG Boost 73.7% 1.42 ± 0.12 FIA = Freund'sIncomplete AdjuvantFIA=Freund's Incomplete AdjuvantIP Challenge of Swiss Outbred CDl Mice

TABLE 6 Dose Dependence of Immunoprotection of Swiss Outbred CD1 Micefrom Orientia tsutsugamushi Kato Strain with Kato r56 Vaccine in thepresence of Freund's Incomplete Adjuvant. Vaccine (Kato r56) Protection0.0 ug  0% 0.8 ug 14% 2.5 ug 43% 8.0 ug 43%  25 ug 57%IP challenge of Swiss Outbred CDl Mice

TABLE 7 Efficacy of the trivalent vaccine (KpKtGm r56) againsthomologous challenge of Kato strain. Challenge Strain of Vaccinated MiceUnvaccinated Mice O. tsutsugamushi (#survived/total#) (#survived/total#)PBS 7/7 7/7 Kato(1,000 LD50;IP)  4/7* 0/7 *Time to death was increasedslightly by vaccination when compared to unvaccinated (PBS injected)control mice.IP challenge of Swiss outbred CDl mice.

The inventors have disclosed efficacy data which supports the use ofone, two or all three antigens in a monovalent, bivalent and trivalentpharmaceutical composition, immunogenic composition and vaccine,respectively. One of ordinary skill in the art will readily recognizethat DNA only approach, a protein only approach, or a prime-boostapproach using DNA in the initial dose and protein in the following dosemay be used for the vaccine.

It is contemplated by the inventor(s) that the following five (5)categories of bioactive substances, combinations thereof and their useare within the scope of this invention:

-   -   1. r56 Karp prepared by PROCEDURE I and II    -   2. r56 Karp prepared by PROCEDURE I and II in combination with        r56 Kato prepared by PROCEDURE I and II    -   3. r56 Karp prepared by PROCEDURE I and II in combination with        r56 Gilliam prepared by PROCEDURE I and II    -   4. rKarp prepared by PROCEDURE I and II,        -   rKato prepared by PROCEDURE I and II,        -   rGilliam prepared by PROCEDURE I and II, and    -   5. Each of the categories (1-4)herein above in combination with        other bioactive and pharmaceutically-acceptable.

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The 56-kilodalton major protein antigen of    Rickettsia tsutsugamushi: molecular cloning and sequence analysis of    the sta56 gene and precise identification of a strain-specific    epitope. Infect. Immun. 58(7):2076-2084.-   35. Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7    RNA polymerase to direct selective high-level expression of cloned    genes. J. Mol. Biol. 189:113-130.-   36. Sugita, Y., T. Nagatani, K Okuda, Y. Yoshida, and H.    Nakajima. 1992. Diagnosis of typhus infection with Rickettsia    tsutsugamushi by polymerase chain reaction. J. Med. Microbiol.    37:357-360.-   37. Suto, T. 1980. Rapid serological diagnosis of tsutsugamushi    disease employing the immuno-peroxidase reaction with cell cultured    rickettsia. Clin. Virol. 8:425-   38. Suwanabun, N., C. Chouriyagune, C. Eamsila, P.    Watcharapichat, G. A. Dasch, R. S. Howard, and D. J. Kelly. 1997.    Evaluation of an enzyme-linked immunosorbent assay in Thai scrub    typhus patients. Am. J. Trop. Med. Hyg. 56:38-43-   39. Tamura, A., N. Ohashi, Y. Koyama, M. Fukuhara, F. Kawamori, M.    Otsuru, P-F. Wu, and S-Y. Lin. 1997. Characterization of Orientia    tsutsugamushi isolated in Taiwan by immunofluorescence and    restriction fragment length polymorphism analyses. FEMS Microbiol.    Lett. 150:225-231.-   40. Urakami, H., S. Yamamoto, T. Tsuruhara, N. Ohashi, and A.    Tamura. 1989. Serodiagnosis of scrub typhus with antigens    immobilized on nitrocellulose sheet. J. Clin. Microbiol.    27:1841-1846.-   41. Weddle, J. R., T. C. Chan, K. Thompson, H. Paxton, D. J.    Kelly, G. Dasch, and D. Strickman. 1995. Effectiveness of a dot-blot    immunoassay of anti-Rickettsia tsutsugamushi antibodies for    serologic analysis of scrub typhus. Am. J. Trop. Med. Hyg. 53:43-46.-   42. Yamamoto, S., N. Kawabata, A. Tamura, H. Urakami, N. Ohashi, M.    Murata, Y. Yoshida, and A. Kawamura, Jr. 1986. Immunological    properties of Rickettsia tsutsugamushi, Kawasaki strain, isolated    from a patient in Kyushu. Microbiol. Immunol. 30:611-620.-   43. Yamamoto, S., and Y. Minamishima. 1982. Serodiagnosis of    tsutsugamushi fever (scrub typhus) by the indirect immunoperoxidase    technique. J. Clin. Microbiol. 15:1128-1132.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is therefore to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described. It iscontemplated that this invention can be used to develop and/or augmentvaccine therapy, prophylactic and therapeutic treatments for otherdiseases caused by facultative intracellular pathogens and/or agentssuch as a virus, bacteria, fungus, venom, pollen, protozoal, andmixtures thereof.

-   Reference: Ohashi, N., H. Nashimoto, H. Ikeda, and A. Tamura 1990.    Cloning and sequencing of the gene (tsg56) encoding a type-specific    antigen from Rickettsia tsutsugamushi. Gene, 91, 119-122

The polypeptide from amino acid 81-448 of the intact protein SEQ ID NO.:5 was cloned into PET24a expression vector. The total # of amino acidsare 368.

>Gilliam sequence number 25 524 aa MKKIMLIASA MSALSLPFSA SAIELGEEGGLECGPYGKVG IVGGMITGAE STRLDSTDSE GKKHLSLTTG LPFGGTLAAG MTIAPGFRAELGVMYLRNIS AEVEVGKGKV DSKGEIKADS GGGTDTPIRK RFKLTPPQPT IMPISIADRDVGVDTDILAQ AAAGQPQLTV EQRAADRIAW LKNYAGIDYM VPDPQNPNAR VINPVLLNITQGPPNVQPRP RQNLDILDHG QWRHLVVGVT ALSHANKPSV TPVKVLSDKI TKIYSDIKPFADIAGIDVPD TGLPNSASVE QIQSKMQELN DVLEDLRDSF DGYMGNAFAN QIQLNFVMPQQAQQQQGQGQ QQQAQATAQE AVAAAAVRLL NGNDQIAQLY KDLVKLQRHA GVKKAMEKLAAQQEEDAKNQ GEGDCKQQQG ASEKSKEGKG KETEFDLSMI VGQVKLYADL FTTESFSIYAGVGAGLAHTY GKIDDKDIKG HTGMVASGAL GVAINAAEGV YVDLEGSYMH SFSKIEEKYSINPLMASVGV RYNFGilliam Strain

1. Primer 5GFGm (784/819) 36 bp Primer: 5′ T T A G C T G C G C.dwnarw.AT A T G A C A A T T G C A C C A G G A T T T A G A 3′ Nde I (SEQ ID NO.6) 2. Primer 56RGm (1929/1894) 36 bp Primer: 5′ A T G A G C T A A C C CG.dwnarw.G A T C C A A C A C C A G C C T A T A T T G A 3′ BamH I (SEQ IDNO. 7)Reference: Tamura, A., H. Ikeda, H. Nashimoto, and N. Ohashi. 1992.Diversity of immunodominant 56-kDa type-specific antigen(TSA) ofrickettsia tsutsugamushi: Sequence and comparative analysis of the genesencoding TSA homologues from four antigenic variants. J. Biol. Chem.267, 12728-12735

The polypeptide from amino acid 81-453 of the intact protein SEQ ID NO.:4 was cloned into PET24a expression vector. The total # of amino acidsare 373.

>Kato sequence number 21 529 aa MKKTMLIASA MSALSLPFSA SAIELGDEGGLECGPYAKVG VVGGMITGVE STRLDPADAG GKKQLPLTTS MPFGGTLAAG MTIAPGFRAELGVMYLANVK AEVESGKTGS DADIRSGADS PMPQRYKLTP PQPTIMPISI ADRDLGVDIPNVPQGGANHL GDNLGANDIR RADDRITWLK NYAGVDYMVP DPNNPQARIV NPVLLNIPQGPPNANPRQAM QPCSILNEDH WRHLVVGITA MSNANKPSVS PIKVLSEKIV QIYRDVKPFARVAGIEVPSD PLPNSASVEQ IQNKMQELND ILDEIRDSFD GCIGGNAFAN QIQLNFRIPQAQQQGQGQQQ QQAQATAQEA AAAAAVRVLN NNDQIIKLYK DLVKLKRHAG IKKAMEELAAQDGGCNGGGD NKKKRGASED SDAGGASKGG KGKETKETEF DLSMIVGQVK LYADLFTTESFSIYAGLGAG LAYTSGKIDG VDIKANTGMV ASGALGVAIN AAEGVYVDTE GSYMHSFSKIEEKYSINPLM ASFGVRYNFKato Strain

1. Primer 56FKt (785/820) 36 bp Primer: 5′ T T A G C T G C A C.dwnarw.AT A T G A C A A T C G C G C C A G G A T T T A G A 3′ NdeI (SEQ ID NO. 8)2. Primer 56RKt (1945/1910) 36 bp Primer: 5′A T A A G C T A A C C C G GA T C C A A G A C C A G C C T A T A T T G A 3′ (SEQ ID NO. 9)Karp: Open Reading Frame (#556-#2151) and Cloned Sequence for ProteinExpression (#793-#1923) (SEQ ID NO. 10)

556 ATGAA AAAAATTATG TTAATTGCTA GTGCAATGTC TGCGTTGTCG 601 TTGCCATTTTCAGCTAGTGC AATAGAATTG GGGGAAGAAG GATTAGAGTG TGGTCCTTAT 661 GCTAAAGTTGGAGTTGTTGG AGGAATGATT ACTGGCGTAG AATCTGCTCG CTTGGATCCA 721 GCTGATGCTGAAGGCAAAAA ACACTTGTCA TTAACAAATG GGCTGCCATT TGGTGGAACG 781 TTGGCTGCAGGTATGACAAT CGCTCCAGGA TTTAGAGCAG AGATAGGTGT TATGTACCTT 841 ACAAATATAACTGCTCAGGT TGAAGAAGGT AAAGTTAAGG CAGATTCTGT AGGTGAGACA 901 AAGGCAGATTCTGTAGGTGG GAAAGATGCT CCTATACGTA AGCGGTTTAA ACTTACACCT 961 CCTCAGCCTACTATAATGCC TATAAGTATA GCTGTACGTG ACTTTGGGAT TGATATTCCT 1021 AACCAGACCTCAGCAGCAAG CACAAGCCGC AGCCTCAGGC TTAATGATGA GCAACGTGCT 1081 GCAGCTAGGATCGCTTGGTT AAAGAATTGT GCTGGTATTG ACTATAGGGT AAAAAACCCT 1141 AATGATCCTAATGGGCCTAT GGTTATAAAT CCGATATTGT TAAATATTCC ACAGGGTAAC 1201 CCTAATCCTGTTGGAAATCC ACCGCAGCGA GCAAATCCGC CTGCAGGTTT TGCGATACAT 1261 AACCATGAGCAATGGAGGCA TTTGGTAGTT GGGCTTGCTG CATTATCAAA TGCTAATAAA 1321 CCTAGCGCTTCTCCTGTCAA AGTATTAAGT GATAAAATTA CTCAGATATA TAGTGATATA 1381 AAGCATTTGGCTGATATAGC TGGTATTGAT GTTCCTGATA CTAGTTTGCC TAATAGTGCA 1441 TCTGTCGAACAGATACAGAA TAAAATGCAA GAATTAAACG ATCTATTGGA AGAGCTCAGA 1501 GAATCTTTTGATGGGTATCT TGGTGGTAAT GCTTTTGCTA ATCAGATACA GTTGAATTTT 1561 GTCATGCCGCAGCAAGCACA GCAGCAGGGG CAAGGGCAGC AACAGCAAGC TCAAGCTACA 1621 GCGCAAGAAGCAGTAGCAGC AGCAGCTGTT AGGCTTTTAA ATGGCAATGA TCAGATTGCG 1681 CAGTTATATAAAGATCTTGT TAAATTGCAG CGTCATGCAG GAATTAAGAA AGCGATGGAA 1741 AAATTAGCTGCCCAACAAGA AGAAGATGCA AAGAATCAAG GTGAAGGTGA CTGCAAGCAG 1801 CAACAAGGAACATCTGAAAA ATCTAAAAAA GGAAAAGACA AAGAGGCAGA GTTTGATCTG 1861 AGTATGATTGTCGGCCAAGT TAAACTCTAT GCTGACGTAA TGATAACTGA ATCAGTCTCA 1921 ATATATGCTGGTGTTGGTGC AGGGTTAGCT TATACTTCTG GAAAAATAGA TAATAAGGAT 1981 ATTAAAGGGCATACAGGCAT GGTTGCATCA GGAGCACTTG GTGTAGCAAT TAATGCTGCT 2041 GAAGGTGTGTATGTGGACAT AGAAGGTAGT TATATGTACT CATTCAGTAA AATAGAAGAG 2101 AAGTATTCAATAAATCCTCT TATGGCAAGT GTAAGTGTAC GCTATAACTT C

The polypeptide from Amino Acid 80-456 of the intact protein SEQ IDNO.: 1) was cloned into PET 24a expression vector. The total number ofAmino Acids are 377.

MTIAPGFRAEIGVMYLTNITAQVEEGKVKADSVGETKADSVGGKDAPIRKRFKLTPPQPTIMPISIADRDFGIDIPNIPQQQAQAAQPQLNDEQRAAARIAWLKNCAGIDYRVKNPNDPNGPMVINPILLNIPQGNPNPVGNPPQRANPPAGFAIHNHEQWRHLVVGLAALSNANKPSASPVKVLSDKITQIYSDIKHLADIAGIDVPDTSLPNSASVEQIQNKMQELNDLLEELRESFDGYLGGNAFANQIQLNFVMPQQAQQQGQGQQQQAQATAQEAVAAAAVRLINGNDQIAQLYKDLVKLQRHAGIKKAMEKLAAQQEEDAKNQGEGDCKQQQGTSEKSKKGKDKEAEFDLSMIVGQVKLYADVMITESVSIKato: Open Reading Frame (#557-2143) (SEQ ID NO. 11) and Cloned Regionfor Protein Expression(#797-1915) is Bolded

557 ATGA AAAAAATTAT GTTAATTGCT AGTGCAATGT CTGCATTGTC 601 ATTGCCGTTTTCAGCTAGTG CGATAGAATT GGGGGATGAA GGAGGATTAG AGTGTGGTCC 661 TTATGCTAAAGTTGGAGTCG TTGGAGGAAT GATTACTGGC GTAGAATCTA CTCGCTTGGA 721 TCCAGCTGATGCTGGTGGCA AAAAACAATT GCCATTAACA ACCTCGATGC CATTTGGTGG 781 TACATTAGCTGCAGGTATGA CAATCGCGCC AGGATTTAGA GCAGAGCTAG GGGTTATGTA 841 CCTTGCGAATGTAAAAGCAG AGGTGGAATC AGGTAAAACT GGCTCTGATG CTGATATTAG 901 ATCTGGTGCAGATTCTCCTA TGCCTCAGCG GTATAAACTT ACACCACCTC AGCCTACTAT 961 AATGCCTATAAGTATTGCGG ATCGTGACCT TGGGGTTGAT ATTCCTAACG TACCTCAAGG 1021 AGGAGCTAATCACCTGGGTG ATAACCTTGG TGCTAATGAT ATTCGGCGTG CTGACGATAG 1081 GATCACTTGGTTGAAGAATT ATGCTGGTGT TGACTATATG GTTCCAGATC CTAATAATCC 1141 TCAGGCTAGAATTGTAAATC CAGTGCTATT AAATATTCCT CAAGGTCCGC CTAATGCAAA 1201 TCCTAGACAAGCTATGCAAC CTTGTAGTAT ACTTAACCAT GATCACTGGA GGCATCTTGT 1261 AGTTGGTATTACTGCAATGT CAAATGCTAA TAAACCTAGC GTTTCTCCTA TCAAAGTATT 1321 AAGTGAAAAAATTGTCCAGA TATATCGTGA TGTGAAGCCG TTTGCTAGAG TAGCTGGTAT 1381 TGAAGTTCCTAGTGATCCTT TGCCTAATAG TGCATCTGTT GAGCAGATAC AGAATAAAAT 1441 GCAAGAATTAAATGATATAT TGGATGAGAT CAGAGATTCT TTTGACGGGT GTATTGGTGG 1501 TAATGCTTTCGCTAATCAGA TACAGTTGAA TTTTCGCATT CCGCAAGCAC AGCAGCAGGG 1561 GCAAGGGCAGCAACAGCAGC AAGCTCAAGC TACAGCGCAA GAAGCAGCAG CGGCAGCAGC 1621 TGTTAGGGTTTTAAATAACA ATGATCAGAT TATAAAGTTA TATAAAGATC TTGTTAAATT 1681 GAAGCGTCATGCAGGAATTA AAAAAGCTAT GGAAGAATTG GCTGCTCAAG ACGGAGGTTG 1741 TAATGGAGGTGGTGATAATA AGAAGAAGCG AGGAGCATCT GAAGACTCTG ATGCAGGAGG 1801 TGCTTCTAAAGGAGGGAAAG GCAAAGAAAC AAAAGAAACA GAGTTTGATC TGAGTATGAT 1861 TGTCGGCCAAGTTAAACTCT ATGCTGACTT ATTTACAACT GAATCATTCT CAATATATGC 1921 TGGTCTTGGTGCAGGGTTAG CTTATACTTC TGGAAAAATA GATGGTGTGG ACATTAAAGC 1981 TAATACTGGTATGGTTGCAT CAGGAGCACT TGGTGTAGCA ATTAATGCTG CTGAGGGTGT 2041 GTATGTGGACATAGAAGGTA GTTATATGCA TTCATTCAGT AAAATAGAAG AGAAGTATTC 2101 AATAAATCCTCTTATGGCAA GTTTTGGTGT ACGCTATAAC TTC

-   Gilliam: Open Reading Frame (#556-#2127) (SEQ ID NO. 12) and Cloned    Sequence (#796-#1899) is Bolded

ATGAAAAAAATTATGTTAATTGCTAGTGCAATGTCTGCATTGTCATTGCCGTTTTCAGCTAGTGCAATAGAATTGGGTGAGGAAGGAGGATTAGAGTGTGGTCCTTACGGTAAAGTTGGAATCGTTGGAGGAATGATTACTGGTGCAGAATCTACTCGCTTGGATTCAACTGATTCTGAGGGAAAAAAACATTTGTCATTAACAACTGGACTGCCATTTGGTGGTACATTAGCTGCGGGTATGACAATTGCACCAGGATTTAGAGCAGAGCTAGGTGTTATGTACCTTAGAAATATAAGCGCTGAGGTTGAAGTAGGTAAAGGCAAGGTAGATTCTAAAGGTGAGATAAAGGCAGATTCTGGAGGTGGGACAGATACTCCTATACGTAAGCGGTTTAAACTTACACCACCTCAGCCTACTATAATGCCTATAAGTATAGCTGATCGTGATGTGGGGGTTGATACTGATATTCTTGCTCAAGCTGCTGCTGGGCAACCACAGCTTACTGTTGAGCAGCGGGCTGCAGATAGGATTGCTTGGTTGAAGAATTATGCTGGTATTGACTATATGGTCCCAGATCCTCAGAATCCTAATGCTAGAGTTATAAATCCTGTATTGTTAAATATTACTCAAGGGCCACCTAATGTACAGCCTAGACCTCGGCAAAATCTTGACATACTTGACCATGGTCAGTGGAGACATTTGGTAGTTGGTGTTACTGCATTGTCACATGCTAATAAACCTAGCGTTACTCCTGTCAAAGTATTAAGTGACAAAATTACTAAGATATATAGTGATATAAAGCCATTTGCTGATATAGCTGGTATTGATGTTCCTGATACTGGTTTGCCTAATAGTGCATCTGTCGAACAGATACAGAGTAAAATGCAAGAATTAAACGATGTATTGGAAGACCTCAGAGATTCTTTTGATGGGTATATGGGTAATGCTTTTGCTAATCAGATACAGTTGAATTTTGTCATGCCGCAGCAAGCACAGCAGCAGCAGGGGCAAGGGCAGCAACAGCAAGCTCAAGCTACAGCGCAAGAAGCAGTAGCAGCAGCAGCTGTTAGGCTTTTAAATGGCAATGATCAGATTGCGCAGTTATATAAAGATCTTGTTAAATTGCAGCGTCATGCAGGAGTTAAGAAAGCCATGGAAAAATTAGCTGCCCAACAAGAAGAAGATGCAAAGAATCAAGGTGAAGGTGACTGTAAGCAGCAACAAGGAGCATCTGAAAAATCTAAAGAAGGAAAAGGCAAAGAAACAGAGTTTGATCTGAGTATGATTGTTGGCCAAGTTAAACTCTATGCTGACTTATTTACAACTGAATCATTCTCAATATATGCTGGTGTTGGTGCAGGGTTAGCTCATACTTATGGAAAAATAGATGATAAGGATATTAAAGGGCATACAGGCATGGTTGCATCAGGAGCACTTGGTGTAGCAATTAATGCTGCTGAGGGTGTATATGTGGACTTAGAAGGTAGTTATATGCACTCATTCAGTAAAATAGAAGAGAAGTATTCAATAAATCCTCTTATGGCAAGTGTAGGTGTACGCTATAACTTC

1. An assay for detecting antibody to scrub typhus comprising: a) Obtaining a sample from a subject; and b) Exposing the sample to at least one polypeptide, selected from the group consisting of: an isolated polypeptide consisting of SEQ ID NO: 4, an isolated polypeptide consisting of SEQ ID NO: 5, or a combination thereof, said polypeptide or polypeptides being the refolded expressed product or products of truncated non-fusion r56 genes from Orientia tsutsugamushi; c) Incubating said sample, wherein said antibody binds said at least one polypeptide forming a complex; d) Binding a detectable label to said complex wherein a detectable signal is produced; e) Detecting the signal, wherein the signal indicates the presence of said antibody.
 2. The assay of claim 1, wherein said assay is selected from the group consisting of Elisa plates, dot-blot matrices, and hand held chromatographic and flow through assay devices.
 3. An assay for detecting antibody to scrub typhus comprising: a) Obtaining a sample from a subject; and b) Exposing the sample to a combination of isolated polypeptides having the amino acids sequences set forth in SEQ ID NO:1, SEQ ID NO: 4, and SEQ ID NO: 5, said polypeptides being the refolded expressed product or products of truncated non-fusion r56 genes from Orientia tsutsugamushi; c) Incubating said sample, wherein said antibody binds said at least one polypeptide forming a complex; d) Binding a detectable label to said complex wherein a detectable signal is produced; e) Detecting the signal, wherein the signal indicates the presence of said antibody.
 4. The assay of claim 1, wherein said assay is selected from the group consisting of Elisa plates, dot-blot matrices, and hand held chromatographic and flow through assay devices. 